Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome

Abstract

The model archaeon Pyrococcus furiosus grows optimally near 100°C on carbohydrates and peptides. Its genome sequence (NCBI) was determined 12 years ago. A genetically tractable strain, COM1, was very recently reported, and here we describe its genome sequence. Of 1,909,827 bp in size, it is 1,571 bp longer (0.1%) than the reference NCBI sequence. The COM1 genome contains numerous chromosomal rearrangements, deletions, and single base changes. COM1 also has 45 full or partial insertion sequences (ISs) compared to 35 in the reference NCBI strain, and these have resulted in the direct deletion or insertional inactivation of 13 genes. Another seven genes were affected by chromosomal deletions and are predicted to be nonfunctional. In addition, the amino acid sequences of another 102 of the 2,134 predicted gene products are different in COM1. These changes potentially impact various cellular functions, including carbohydrate, peptide, and nucleotide metabolism; DNA repair; CRISPRassociated defense; transcriptional regulation; membrane transport; and growth at 72°C. For example, the IS-mediated inactivation of riboflavin synthase in COM1 resulted in a riboflavin requirement for growth. Nevertheless, COM1 grew on cellobiose, malto-oligosaccharides, and peptides in complex and minimal media at 98 and 72°C to the same extent as did both its parent strain and a new culture collection strain (DSMZ 3638). This was in spite of COM1 lacking several metabolic enzymes, including nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase and beta-glucosidase. The P. furiosus genome is therefore of high plasticity, and the availability of the COM1 sequence will be critical for the future studies of this model hyperthermophile.

Mannosylglycerate and Di- myo -Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

“…Although both strains showed similar growth rates over the 88 to 98°C temperature range, the temperature for the maximum cell density of COM1 was 90°C and growth was not observed at 100°C (after 8 h), while the temperature for the maximum cell density of the wildtype strain was 95°C and some growth was observed at 100°C (7, 8). The explanation for the observed phenotypic differences could be related to the large number of changes encountered at the genome level (14). Exploring these differences by complementing the COM1 strain with genes that are functional in the DSM 3638…”

“…The genetic system is based on a variant of P. furiosus DSM 3638 T designated COM1, which is highly competent for external DNA uptake and recombination. The genome sequence of strain COM1 showed extensive changes; however, the major metabolic features of the wild-type strain appear to be conserved (14).…”

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

“…The genetically tractable P. furiosus strain, COM1, has been used for making gene deletions (14 -16), overproducing its proteins (17,18), and for metabolic engineering (9,19). The COM1 strain is remarkably efficient at both DNA uptake and recombination (13,20,21), although the factors responsible are not clear (22). It is also not known how large of a DNA fragment can be functionally inserted into the P. furiosus chromosome.…”

The Order Thermococcales and the Family Thermococcaceae

“…These three Pyrococcus species have relatively large sections of synteny, but only short stretches of homologous regions can be identified when comparing these to the Thermococcus species T. gammatolerans, T. kodakarensis, and T. onnurineus (Zivanovic et al 2009). The genome sequence of a competent strain of P. furiosus, COM1, contains numerous chromosomal rearrangements, deletions, and single base changes compared to the reference genome, and more than 100 of the 2,134 predicted genes showed differences among the two strains (Bridger et al 2012). Notably, the COM1 genome contained 39 full insertion elements compared to 29 in the reference genome, suggesting these may have played a role in ''laboratory evolution'' of this strain.…”

“…Over nearly three decades or so, P. furiosus has been one of the best studied of all of the so-called hyperthermophilic species, which are usually defined as organisms having an optimum growth temperature of at least 80 C (Stetter 2006). The recent characterization of a competent strain of P. furiosus that can be metabolically engineered for applied purposes (Basen et al 2012;Bridger et al 2012;Lipscomb et al 2011) promises to accelerate and broaden studies of this organism. The genus Palaeococcus is an underrepresented genus within this family and is so far not extensively studied.…”